In a clinical chemistry test and a clinical immunologic test, many of rapid tests for qualitative and quantitative diagnosis have been developed in the field of a hospital-related examination using blood as a sample.
Though part of the clinical tests can be carried out on whole blood, it is necessary to use plasma or serum isolated from a blood sample in order to attain a correct result of the test. If not isolated, red blood cells or an impurity in a blood sample blocks reflected light, transmitted light and emission measured in the test, affecting the measurement adversely.
Measurement on the kind and concentration of a blood component in a clinical laboratory test is usually performed such that whole blood is sampled with a blood collecting device, the sampled whole blood in a blood sampling tube is centrifuged to obtain serum or plasma and the obtained serum or plasma is used as a test sample.
Centrifugation, however, requires hundreds of μL or more of the whole blood amount and further consumes labor and time. Especially, in many fields of biochemical examinations, the presence of blood cells such as red blood cells disturbs the examinations; therefore, the serum or plasma is isolated from the blood in advance and used as a test sample. Hence, a necessity arises for a process in which blood sampled from a patient or a subject is at first coagulated in advance of an examination and then centrifuged to thereby obtain serum. While in Clinical Chemistry Analyzers available in recent years, a necessary mount of a test sample used in any of most examinations is as small as in the range of from several μL to tens of μL because of advancement in technology, a sampled blood amount depends on specifications of a centrifuge and a size of a blood sampling tube, which wastefully requires a blood amount more than necessary.
In the conventional centrifugal separation method, with a smaller test sample amount, separated red blood cells are re-suspended in plasma or serum; a problem arises that it is difficult to obtain only plasma or serum. therefore, in the centrifugal separation method, a test sample amount has to be in the range of from hundreds of μL to several mL, which necessitates a syringe to sample blood, having led to a fault that a great burden is imposed on a person to be examined. Furthermore, since the centrifugal separation method requires a special apparatus for centrifuging and is carried out only in batches, it is disadvantageous in cost for treatments on small number of test samples and unsuitable for an on-demand and instant treatment.
A method of measuring a bilirubin concentration of a neonate is such that a blood-sampling needle is stuck in a sole of the neonate to sample blood with a capillary tube. Sampled blood is separated to obtain serum or plasma by a centrifuge for capillaries and a bilirubin concentration is attained by conversion of an absorbance in the proximity of a wavelength in the range of from 450 to 460 nm, which is a bilirubin absorption wave length, measured with a photometer dedicated thereto as shown in FIG. 17. Therefore, in order to perform the examination, a centrifuge for exclusive use has been required and a time has been consumed in separating operation longer than in measurement itself.
In FIG. 17, reference numeral 50 designates a light source; 52 , a optical light chopper; 54, a heat absorbing filter; 56, a lens; 58, a filter disk; 60a and 60b, interference filters; 62, a capillary tube; 64, a capillary tube holder; 66, a photodiode; 68, an amplifier; and 70, a meter. The capillary tube 62 in which plasma or serum isolated from blood is accommodated is to be held on the capillary tube holder 64 and light from the light source 50 is to be passed through the optical light chopper 52, the heat absorbing filter 54, the lens 56 and the interference filters 60a and 60b and is irradiated to the capillary tube 62, thereby an absorbance being measured at a wavelength in the proximity of a value in the range of from 450 to 460 nm with the photodiode 66 to detect a bilirubin concentration with the meter 70.
As a means to solve the above problem, there has been known dry chemistry. This test is conducted such that a small amount of blood is dropped onto a plate constructed with a plasma or serum separation layer, which is a fiber filter such as a filter made of glass fibers; and a reaction layer located in the lower layer, and then plasma or serum is isolated in the plasma or serum separation layer and a reaction and a color development occur in the lower layer, which is subjected to colorimetry. The dry chemistry is a simple and convenient method not requiring troublesome sampling of plasma or serum with a centrifuge, whereas the test can be applied only to a system foe exclusive use, in which the plasma or serum separation layer and the reaction layer is integrated into one piece. Since one plate can be used to measure only one examination item, plural plates are required for examining plural items; therefore, the dry chemistry is too expensive for its simplicity and convenience to be used widely.
Proposals have been made on methods each obtaining plasma or serum from blood without using a centrifuge (JP-A Nos. 53-72691, 60-11166 and the like). These methods have problems that separation is time consuming, red blood cells cause to clog and hemolysis occurs; they have not been put into practical use.
Various proposals have been made on a technique in which blood is flown through a layer made of a fiber filter under pressure to thereby isolate plasma or serum. Methods and tools disclosed in JP-A Nos. 61-38608, 4-208856 and 5-196620 can sample plasma or serum without using a centrifuge, whereas an amount of plasma or serum obtained is small. Consequently, the techniques have not been widely applied.
A method in which a conventional plasma or serum separating filter is employed has a problem that a special device or tool is required for pressurization or reducing a pressure for collecting plasma or serum and operations are troublesome.
As described above, there has not been available, in the current state, a tool exhibiting a sufficient performance in isolating a plasma or serum component from a small amount of blood to be used for a clinical laboratory test in a short time with good efficiency.
On the other hand, as described above, the following matters may be mentioned in the centrifugal separation method; it takes too much time for separation; it is difficult to sample serum or plasma after separation from a small amount of a whole blood sample; and a necessity arises for an auxiliary facility that is not generally usable outside of an examination room. For the reasons, the centrifugal separation method is perfectly improper for a test implemented by an expert except a clinical laboratory test engineer at a site, other than an examination room, in a point of care test, which is performed in a bed-side test, an emergency test and the like.
In order to avoid the problems, many techniques have been devised and proposed. In these techniques, generally many kinds of materials have been employed for fabricating a filter. As filter materials, there have been conventionally known film materials such as paper, textile, glass, synthetic fibers or the like, each of which has proper pore sizes.
For example, a method has been disclosed in U.S. Pat. No. 4,816,224, in which glass fiber filter paper and a porous film with a specific size are laminated. U.S. Pat. No. 4,753,776 is related to a device for separating plasma or serum from red blood cells by bringing whole blood into contact with a filter made of glass fibers capable of carrying a red blood cell aggregating agent, and teaches a method in which a thickness and diameter of the glass fiber filter is defined and there is fed to a capillary tube flow device plasma or serum isolated by capillary force from whole blood while the whole blood flows through the filter. In both methods, a practical isolated amount of plasma or serum is usually 25% or less, which causes clogging with ease.
Then, in U.S. Pat. No. 5,665,238, there has been disclosed a method in which serum is isolated with a layer made of a fiber filter such as a glass fiber filter having a pore size of 3 μm or less, and containing mannitol which is a polysaccharide at a content in the range of 1 to 40% and albumin at a content in the range of 0.1 to 15% to thereby achieve separation with a high yield, but this method has faults that no consideration is given to hemolysis and a time taken for separation is long.
In U.S. Pat. No. 3,146,163 and DE-A-1498577, another method is described in which whole blood is applied to a material coated with a red blood cell aggregating agent such as a vegetative red blood cell aggregating agent in order to isolate plasma or serum, and as carrier materials that can be used, there are named plastic and fibrous materials such as a card board.
In JP-A No. 61-118661, there has been disclosed a separation method for whole blood in which whole blood is treated with a matrix impregnated with lectin to isolate plasma or serum, wherein the matrix is an absorbing material having a relative resistance as high as 40% to a fluid flow. Furthermore, there are shown matrices each having a fibrous structure or a fiber structure, and having a resistance as small as possible to a fluid flow, and as preferable materials, there are exemplified cotton, viscose fibers and a cellulose material. An apparent fault of this method, however, is a necessity for washing separated plasma or serum from the matrix with a diluent.
In JP-A No. 64-21362, there has been disclosed still another apparatus isolating plasma or serum from whole blood. The apparatus contains an absorbing matrix having treated with a reagent aggregating blood cells, and thrombin or lectin is named as the reagent aggregating blood cells. As absorbing matrices, there are recommended hydrophobic powder, sponge, clay, fibers and a polymer, and fiber-containing paper is recognized as a polymer and a filter paper is a preferable matrix.
JP-A No. 61-207966 teaches a reagent for isolating plasma or serum from whole blood in a case where an absorbing matrix impregnated with lectin is used as a separation layer.
JP-A No. 2-140147 discloses that polycation is fixed on an absorbing solid material so as to create a cationic surface to which red blood cells are coupled when being brought into contact with whole blood, and paper is named as a preferable carrier material.
JP-A No. 60-36961 discloses that plasma or serum is isolated from whole blood using an absorbing porous material impregnated with a material having some polar groups. Examples of absorbing porous materials generally include paper, pad and fibers.
EP-A-0305803 discloses an apparatus for isolating red blood cells from a red blood cell containing body fluid, which comprises a fiber-containing filtration layer including a red blood cell aggregating antibody and if desired, lectin or a glass fiber filtration layer and lectin.
Of the known whole blood separating methods described above, especially practically effective is a method in which whole blood is brought into contact with a fiber containing layer having a red blood aggregating function to cause the fiber containing layer to hold red blood cells. A problem encountered in a case where a fiber-containing layer of this kind is employed is that hemolysis occurs if red blood cells are brought into contact with a fiber-containing layer.
On the other hand, a method disclosed in U.S. Pat. No. 5,262,067 is a good method in which a glass fiber layer containing a red blood cell aggregating material is coated with polyvinyl alcohol or polyvinyl alcohol/polyvinyl acetate to thereby enable hemolysis to be suppressed so as to give no practical influence on a plasma or serum component isolated from whole blood, but this method has a problem to take a long time for separation.
Hemolysis releases hemoglobin from a red blood cell to thereby decolorize plasma or serum. Since such decolorization can considerably disturb a colorimetric analysis, as hemolysis of red blood cells caused by glass is smaller in amount, a less influence of an inhibitor is exerted on measurement.
The present invention has been made in light of the above problems and it is an object of the present invention to provide a plasma or serum separator and a plasma or serum sampling method capable of isolating plasma or serum with good efficiency from a small amount of blood without using a centrifuge and without causing leakage of a blood cell component or hemolysis, and in addition, capable of isolating and collecting plasma or serum from a whole blood test sample in a short time with simplicity in a blood test in the scene of medical care requiring an instant treatment any time such as an emergency test, home-use test or the like.
The present inventor has conducted a study on subjects to find a possibility separating a cellular component in non-diluted blood, especially red blood cells from plasma or serum, to prevent hemolysis during the separating, to find a possibility of high speed separation during the separating and to enable the separating in a point of care test in a clinical laboratory test with the result of findings that to the present inventor's surprise, by using a fiber containing a layer including glass fibers coated with hexylene glycol, butoxypropanol or butoxymethylacrylamide as a plasma or serum separation layer, hemolysis does not substantially occur and the fiber containing layer is suited for separating very well a cellular component of non-diluted whole blood from plasma or serum, which has led to the present invention.
It is a second object of the present invention to provide a plasma or serum separating method of isolating-plasma or serum from whole blood, a separator, a test carrier, and glass fibers, in which not only is separation of red blood cells performed in a short time, but a separation ability is also high, and in addition less of hemolysis occurs.